As the nanoparticles carry red fluorescence dye for cell membrane, cellular uptake of PCFMN and FMN can be detected by fluorescence inversion microscope (Olympus, Japan). Chondrocytes were cultured in 24-well plates (1 × 104 cells/well). When cells were fully attached, fresh medium containing PFMN-DID (without cartilage-targeting peptide) (10 μL, 1 mg/mL) and PCFMN-DID (10 μL, 1 mg/mL) fluorescent nanoparticles were added as a substitute for the old medium. After 24 h incubation, cold PBS and 4% paraformaldehyde were respectively used to wash cells and then fix for 20 min. Following, chondrocyte nuclear were stained by DAPI for 15 min. Finally, the plate was analyzed by a fluorescence inversion microscope (Olympus, Japan) to obtain corresponding fluorescence images.
Fluorescence inversion microscope
Manufactured by Olympus
Sourced in Japan
The Fluorescence inversion microscope is a specialized lab equipment designed for imaging and analysis of fluorescent samples. It utilizes an inverted optical configuration to enable observation of specimens from the bottom of a culture dish or plate. The microscope is equipped with illumination and detection systems optimized for fluorescence imaging, allowing researchers to visualize and study the distribution and dynamics of fluorescently labeled cellular structures, molecules, or other biological entities.
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Lab products found in correlation
Matrigel, by BD (5 mentions)
Interleukin 6 (il 6), by Boster Bio (1 mentions)
Mmp 13, by Boster Bio (1 mentions)
Matrigel matrix, by BD (1 mentions)
Transwell chamber, by Corning (1 mentions)
Fluorescent labeled secondary antibody, by Bioss Antibodies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Transwell assay, by Corning (1 mentions)
Hematoxylin and eosin staining, by Solarbio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Phosphorylase inhibitor, by Wuhan Servicebio Technology (1 mentions)
Hif 1a, by Novus Biologicals (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
One step tunel apoptosis assay kit, by Beyotime (1 mentions)
Sds page, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Cell culture insert, by BD (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
23 protocols using fluorescence inversion microscope
1
Fluorescence Imaging of Nanoparticle Uptake
2
Chondrocyte Biomarker Expression Analysis
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The expression of OA catabolic biomarkers IL-6 and MMP-13 in chondrocytes was assessed by immunofluorescence. Chondrocytes were fixed with 95% ethanol for 30 min and permeabilized with 0.1% Triton X-100 for 10 min. Samples were incubated with primary antibody as follows: IL-6 (1:200, Boster, China), and MMP-13 (1:200, Boster) at 4 °C overnight. Then the samples treated with the secondary antibodies FITC-anti-rabbit IgG (1:50, Boster) for 60 min at 37 °C and counterstained with DAPI for 5 min. Finally, the fluorescence images were photographed using a fluorescence inversion microscope (OLYMPUS, Japan).
3
Cell Migration and Invasion Assays
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The wound-healing assay was conducted to evaluate cell migration ability: 3 × 105 cells per well were seeded in triplicate into six-well plates and cultured for 24 h. The cell surface was scratched with a pipette tip. The suspended cells were washed off with PBS and cultured with fresh DMEM medium without serum for an additional 24 h. The area of the cell crawling from the scratch toward the center is observed under a microscope, and the size of the area indicates the cell’s ability to migrate (20 (link)). The migration distance was analyzed by Image-Pro Plus 6.0 software.
Cell invasion assays were performed using Transwell chambers (8-μm pore size; Corning, United States) coated with Matrigel matrix (BD Biosciences, United States). A detailed experiment was performed similar to that previously reported (21 (link)). The invaded cells were counted with a fluorescence inversion microscope (Olympus, Japan) at 200× magnification. Six fields per well were randomly selected under the microscope, and the average number of cells in each field was calculated.
Cell invasion assays were performed using Transwell chambers (8-μm pore size; Corning, United States) coated with Matrigel matrix (BD Biosciences, United States). A detailed experiment was performed similar to that previously reported (21 (link)). The invaded cells were counted with a fluorescence inversion microscope (Olympus, Japan) at 200× magnification. Six fields per well were randomly selected under the microscope, and the average number of cells in each field was calculated.
4
Wound Healing Assay for Cell Migration
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Cell migration was assessed using a wound healing assay. Briefly, cells (3×105 cells/well) were seeded into a 6-well plate and incubated in RPMI-1640 medium containing 10% FBS at 37°C until 80% confluence was reached. Subsequently, cells were serum-starved for 24 h. A straight scratch was then introduced in the cell monolayers using a 200 µl plastic pipette tip. PBS was used to wash the cells and remove any debris. Following incubation for further 48 h in serum-free medium, the average distance of cells migrating into the wound was measured using an fluorescence inversion microscope (Olympus; magnification, 100×).
5
Evaluating Doxorubicin Delivery Vehicles
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MCF-7/Adr cells were seeded in 24-well plates at a density of 1×105 cells/well and incubated for 24 hours. Then, the cells were incubated with DOX-Sol, HA-VES4/DOX, HA-VES7/DOX, and HA-VES12/DOX (8 μg/mL DOX) for 48 hours. For qualitative observation, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and the cellular and nuclear morphology was observed with a fluorescence inversion microscope (Olympus Corporation, Tokyo, Japan). For quantitative measurement, the cells were trypsinized, washed with cold phosphate-buffered saline (PBS) twice, harvested, and stained with Annexin V-FITC and propidium iodide for 10 minutes at room temperature in the dark. Thereafter, the binding buffers were added and the samples were analyzed by fluorescence-activated cell sorting (FACS).
6
Colony Formation Assay
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Cells (300 cells/well) were seeded in six-well plates and cultured in a 37°C 5% CO2 incubator. The culture medium was replaced every 2 days. After 2 weeks, when colony formation became visible, the cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes, followed by staining with 0.5% crystal violet dye (Beyotime, C0121-100mL) for 15 minutes. The colonies were subsequently imaged and counted using a fluorescence inversion microscope (Olympus, Tokyo, Japan) and ImageJ software (version 1.52r).
7
Colony Formation Assay Protocol
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Cells in the logarithmic growth phase were seeded into 6-well plates at the density of 600 cells/well and were cultured at 37°C for 14 days until colony formation became visible. Culture medium was regularly changed every 2 days. The colonies were subsequently fixed with 4% paraformaldehyde for 30 min at room temperature, stained with 0.5% crystal violet for 10–30 min and imaged under a fluorescence inversion microscope (Olympus; magnification, 10×). ImageJ software (version 1.52r; National Institutes of Health) was used to count colonies.
8
Immunofluorescence Analysis of Synovium Samples
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Synovium samples from five patients with OA and five patients with knee joint trauma were harvested and immediately fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and cut into 4 µm-thick serial sections. Next, the sections underwent dewaxing, rehydration, antigen retrieval, and blocking. Subsequently, the sections were stained using TDO2 antibody (1: 400; Proteintech, USA) overnight at 4 °C. After washing with PBS (three times for 5 min), the sections were incubated with the corresponding fluorescent-labeled secondary antibody (Bioss, Beijing, China) for 1 h at 4 °C in the dark and then counterstained with 4',6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Nanjing, China) for 5 min. Images were captured using a fluorescence inversion microscope (Olympus, Japan). Isotype control immunofluorescence with isotype-matched IgG did not show any staining (Supplementary Fig. 1 A).
9
Teratoma Formation from hESCs
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After 5–7 days of culture with eiMEFs, hESCs colonies were suspended with 1mg/ml collagenase IV (Invitrogen, USA) and 1X dispase. Then 5–10×106 hESCs were injected intramuscularly into Severe Combined Immunodeficiency Disease (SCID) mice. Teratomas formed after 2–3 months. Mices were sacrificed and teratoma tissues were dissected into sections and then fixed in 4% PFA. The fixed sections were stained with hematoxylin (Sigma, USA) and eosin (Sigma, USA) and photographed with the microscope of fluorescence inversion microscope (Olympus, Japan). The Institutional Animal Care Committee of Nanjing Medical University approved the experimental protocol.
10
Evaluating TNBC Cell Invasion
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The invasive ability of TNBC cells was evaluated using Transwell assay (pore size, 8.0 µm; Corning Inc.) coated with Matrigel (BD Biosciences) overnight at 37°C. Briefly, 500 µl serum-free media containing 2×105 cells were seeded in the upper chamber, and the lower chamber was filled with media containing 10% FBS. Invasive cells were stained with crystal violet 48 h after incubation, and were subsequently imaged under a fluorescence inversion microscope (Olympus; magnification, 100×). Cell number was analyzed using ImageJ software (version 1.52r; National Institutes of Health).
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